Summary

This case summarizes the use of molecular diagnostics for determining
the pattern of porcine reproductive and respiratory syndrome virus
(PRRSV) transmission in piglets after birth and whether transmission
was related to herd parity. We also assessed the effectiveness
of the recommended intervention strategy in controlling viral
shedding.

Infection of nursery
pigs with porcine reproductive and respiratory syndrome virus
(PRRSV) is associated with an increase in respiratory disease,
poor growth rates, and elevated mortality. Porcine reproductive
and respiratory syndrome is primarily controlled through the use
ofmodified-live virus (MLV) vaccines or management.1,2
As with other infectious agents, it is prudent to vaccinate prior
to exposure. Therefore, it is important to determine when infection
occurs in the life of the pig. A new diagnostic test now available
to detect PRRS viral nucleic acid is the polymerase chain reaction
(PCR). This technique can be conducted on serum, semen, or tissue
to detect viral RNA or DNA. The purpose of this field study was
to describe the application of PCR diagnostics and molecular sequencing
to a case of postweaning PRRS.

Case history

A 300-sow, farrow-to-finish, one-site production system was
first infected with PRRSV in 1995 (Figure
1). After the infection, nursery mortality increased from
an annual rate of 2.0% to 4.5%. An immunization protocol using
RespPRRS vaccine (Boehringer Ingelheim/NOBL Laboratories, St.
Joseph, Missouri) was initiated postinfection: sows were vaccinated
on day 6 of lactation and pigs at weaning (day 16). After vaccination
was initiated, the mortality rate dropped to 3.0%, and remained
at that rate for 2 years.

In January 1997, the gilt development protocol for the herd
was modified (Figure
1). Previously, gilts had been introduced from a PRRSV-negative
source at 6 months of age, and vaccinated 60 days prior to breeding
(7.5-8 months of age). In 1997, gilts began to be introduced as
weaned piglets from the same PRRSV-negative source into a developer
facility and raised to the desired breeding age. Gilts were vaccinated
upon arrival at the developer, at selection (5 months of age),
and again on day 6 of lactation.

From January 1998-July 1998, nursery mortality averaged 4.25%,
and the percentage of poor-doing pigs (culls) at the end of the
nursery period increased from 2% to 10%. Cull animals were in
poor condition, with long hair coats and dyspnea. Over the 12
months immediately prior to the study, the mean PRRSV ELISA S:P
value was 0.79 (SD 0.51, range: 0-2.78).

It was not clear why the mortality rate in this herd remained
elevated and the growth rate did not improve after vaccination
and the new gilt development protocol was initiated. The protocol
of vaccination had not been changed between January 1997 and July
1998, nor had another vaccine been used. Protocol compliance and
vaccine handling were determined to be acceptable.

Diagnostic results from these five piglets raised the possibility
that piglets were being infected with wild-type virus before being
vaccinated. A testing protocol was designed to investigate this
possibility.

Testing protocol

The testing protocol was calculated to detect at least one
infected pig assuming a 10% prevalence of infection with 95% confidence,
requiring a minimum of 30 samples per group sampled. In order
to determine whether infection was occurring during a specific
time in the life of the piglet, 30 samples were collected on lactation
days 1, 2, 4, 6, 8, 10, 12, 14, and 16 (weaning). To assess whether
a specific parity was involved, samples collected per lactation
day were equally distributed over all parities present on the
farm during the study period. Five litters per parity were sampled,
and one piglet was randomly selected from each litter. Cross sectional
sampling strategies were used at all times, and the testing protocol
was repeated three times. In order to maintain litter integrity,
piglets were not cross fostered during the sampling period.

Piglets were serosampled using standard venipuncture. Prior
to sampling, ice packs were added to the blood collection kit
to keep samples cool during the sampling process. Although a refrigerated
centrifuge was not available, sera were immediately cooled to
4 degrees C and shipped to the diagnostic laboratory on ice for
PCR testing using overnight mail.

In order to control cost, two to three sera were pooled within
each parity group. An aliquot (1.0 mL) of each individual sample
was stored at -70 degrees C. If a positive signal was detected
from a pool of sera, the three individual samples that comprised
the pool were individually tested using PCR. Positive samples
were then applied to cell culture for isolation of PRRSV,3
and then sequenced to determine their relationship to the field
strain isolated from the nursery pigs. As a control, a dose of
PRRSV vaccine was taken from the farm and submitted for isolation
and sequencing as well.

A total of 35 samples over seven parities were collected on
each designated lactation day. Despite the presence of clinical
signs in nursery pigs, no detectable clinical abnormalities were
detected in the suckling piglets sampled. Positive PCR signals
were detected during the first and third trial, and only in samples
collected from gilt litters. A total of five of 135 (3.7%) of
the pooled samples collected from gilt litters were positive by
PCR and virus isolation. All isolates were recovered from different
piglets on days 1, 2, and 16. Sequencing indicated that all isolates
were identical to the wild-type strain previously isolated from
lung tissue of nursery pigs. Vaccine virus was not detected during
any of the trials, despite the vaccination of all adult females
at day 6.

Discussion

The TaqMan(TM) PCR test was used throughout this study. The
analytical sensitivity of this assay has been demonstrated to
be between 1.7-170 TCID50 per reaction, and in specificity
testing it detected 18 of 18 North American isolates of PRRSV
(Collins J, personal communication, 1998).

The results of this study are not generalizable to all farms.
Its purpose was to evaluate the usefulness of molecular diagnostics
to determine the pattern of viral infection on a commercial swine
farm, not to promote the use of a specific commercial product,
or to promote standardized administration protocols.

The results from this field study indicate that molecular diagnostics
are useful tools for determining whether suckling piglets are
infected with PRRSV prior to vaccination. In addition, using our
sampling protocol we were able to determine the relationship of
parity and shedding of virus, and the similarities in viral RNA
that existed between isolates recovered from suckling and nursery
pigs. The RNA of these isolates were determined to be not of vaccine
viral origin. The difference of two base pairs detected between
the vaccine virus and its laboratory standard were within normal
variability limits (Collins J, personal communication, 1998).

The primary limitation of the study was our inability to include
controls. Because samples were not collected prior to piglet suckling,
we cannot rule out the possibility that the two piglets detected
positive on days 1 and 2 of age had been infected transplacentally.
Furthermore, we could not determine whether the source of the
virus was colostrum, milk, or horizontal spread from infected
littermates or dams (Wagstrom EA, et al; Proc AASP Ann Meet.
1998; 405-406). Finally, since PCR and sequencing were not available
during the initial outbreak, it was impossible to compare sequences
of PRRSV that originally infected the herd to the isolate we identified
through PCR in this study.

It is interesting that we detected viral shedding only in gilt
litters. One possible explanation for this phenomenon may be that
natural immunity had developed after infection in older sows (parities
two through seven) in contrast to gilts. It is also possible that
immunity induced by the vaccine may endure longer in older sows.The gilt development program adapted in 1997 may also have
contributed to this phenomenon. In this herd, it was necessary
to breed gilts of this genetic line at 7.5-8 months of age, creating
an extended interval between the vaccinations at selection and
during lactation according to this new protocol. The licensure
studies to test the duration of immunity for this vaccine were
terminated at 4 months post vaccination(Polson DD.
RespPRRS: a PRRS Vaccine Review. 1994; 9); it is therefore
unknown whether a prolonged interval between vaccinations would
have a negative impact on the immune status of a large population.
After we completed the testing protocol described above, we began
revaccinating gilts at day 50 of gestation. The use of the commercial
vaccine described in the study constitutes off-label use and requires
a valid veterinary-client-patient relationship.

Since the initial testing protocol we describe in this paper
was conducted, we have continuously monitored this herd using
the same protocol. At the time of this writing, more than 1260
suckling piglets have been tested by PCR across all parities,
and no evidence of PRRSV in suckling piglets has been detected
by PCR in sow or gilt litters. Current production data (1999)indicate that nursery mortality has dropped to 2% and the
percentage of culls has declined to 2.5%.

The phrase "breeding herd stability" has been used
throughout the swine industry to describe the absence of vertical
transmission of PRRSV. Based on previous field experience, using
serology as the primary diagnostic tool, this herd would have
been classified as a stable vaccinated herd.2 However,
the results of the PCR testing in this study indicated that serology
alone may not be adequate to define the PRRSV status of a breeding
herd. This case suggests that molecular techniques can be useful
in conjunction with serology, observations of clinical signs,
and scrutiny of production data in correctly determining the PRRSV
status of a breeding herd and to define and pinpoint the period
in the life of the piglet during which infection seems to be occurring.
Once this information is collected and evaluated, the optimal
intervention strategies can be determined for a herd.

Implications

The measure of breeding herd stability is the absence of
vertical transmission.

Molecular diagnostics such as PCR and sequencing are helpful
for determining the point of PRRSV infection in the life of a
piglet.

PRRSV vaccination strategies should be established on an
individual herd basis.

Parity-specific PRRSV vaccination programs may be necessary
to control PRRSV in herds.

Monitoring the PRRSV status of suckling piglets using a statistically
valid sample size is a crucial component of a PRRS control program.